Cellular aging and senescence of somatic cells and tissues occurs in all animals. In metazoans, aging consists of decreased regenerative capacity of renewable tissues, resulting from altered proliferation and differentiation of tissue stem and progenitor cell populations. In addition, certain model organisms, including the yeast, S. cerevisiae, have yielded remarkable insights into molecular pathways involved in aging and senescence. These include genomic changes such as telomere shortening, altered heterochromatic structure and function, and profound changes in gene expression. Our hypothesis is that epigenetic regulation is likely to be a key determinant underlying these age-associated changes in genomic structure and function. We will focus on two aspects of chromatin regulation: post-translational modifications of histone proteins, and chaperone-mediated assembly of histone variants into the nucleosomal scaffold. We will use several models: replicative lifespan-restriction of budding yeast mother cells and yeast senescence caused by telomere shortening, as well as senescence and differentiation of mammalian bone marrow-derived mesenchymal progenitor cells and aging of bone tissue. The projects are linked through collaborative studies of two major experimental focuses: the histone deacetylase Sir2 function in yeast aging and senescence, and the role of histone chaperones HIRA/ASF1a/UBN1 in regulating bone cell differentiation and senescence. The overarching theme is that aging, senescence and tissue differentiation are related by a strong epigenetic regulatory component. Our combined preliminary data support these aims and offer several unique and synergistic avenues to address major epigenetic questions of cellular and tissue aging. These key questions are (1) Is Sir2 activity, regulation, and localization directly involved in yeast replicative aging and senescence? Is histone H4 K16ac the key substrate of Sir2 related to its role in aging/senescence? (2) Can small molecules that alter Sir2 activity in vitro, alter the kinetics of aging/senescence in vivo? (3) What is the role of HIRA/ASF1a in regulation of chromatin structure during differentiation of bone marrow-derived mesenchymal progenitor cells? (4) What is the structure of the ASF1/HIRA/histone trimeric complex? What is the structure of HIRA in association with the chromatin protein Ubn1? How can these structure/function insights be used to enhance our understanding of cell senescence and tissue aging? Human disease is increasingly being linked to epigenetic pathways. Our project will provide new insights into the molecular basis of epigenetic regulation that drive eukaryotic aging, and will generate novel small molecule regulators of these pathways. Because epigenetic alterations are reversible, these studies have the potential to develop therapeutic agents and targets to alleviate debilitating aspects of aging and age-related diseases.